Composition for Promoting Ketone Body Production

ABSTRACT

An object of the present invention is to provide a novel composition for promoting ketone body production. The present invention provides a composition for use in promoting ketone body production, comprising one or more amino acid(s) selected from the group consisting of citrulline, leucine, cysteine, taurine, glutamine and aspartic acid. The composition according to the present invention may further comprise either or both of a medium-chain fatty acid and a medium-chain fatty acid ester.

CROSS-REFERENCE TO RELATED APPLICATION

The present application enjoys the benefit of priority from the priorJapanese Patent Application No. 2016-161267 filed on Aug. 19, 2016, theentire disclosures of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a composition for use in promotingketone body production.

BACKGROUND ART

The “ketone body” is a generic term for acetoacetic acid,β-hydroxybutyric acid and acetone. Normally, fatty acids are β-oxidizedin the liver to form acetyl CoA, which is completely oxidized in the TCAcycle. On the other hand, lack of carbohydrates necessary for the TCAcycle causes insufficient rotation of the TCA cycle so that acetyl CoAis accumulated and converted into acetoacetic acid via acetoacetyl CoA.Acetoacetic acid is decarbonized into acetone and enzymatically reducedinto β-hydroxybutyric acid. Due to the reduction in glucose in thetissue caused by diabetes and the lack of carbohydrates caused byfasting, the ketone body production in the liver increases, and theketone bodies are utilized as an energy source. Similarly to glucose andthe like, the ketone bodies are utilized as an energy source in manytissues. Especially, the brain utilizes only glucose and ketone bodiesas energy sources. When glucose is depleted, the ketone bodies serve asthe only energy source and thus play an important role.

It has become clear that ketogenic diet, which is a dietary regimeninvolving production of a lot of ketone bodies in a living body, isuseful for prevention, treatment and the like of various diseases.Ketone diet is a high-fat low-carbohydrate diet in which ketone bodyproduction is promoted by extreme decrease in carbohydrate intake andintake of a lot of fat. Ketogenic diet is known to be useful for thetreatment of intractable epilepsy and GLUT1 deficiency and thesuppression of seizures of intractable epilepsy, and, more recently, hasbeen reported to be useful for the prevention and treatment ofneurodegenerative diseases such as Alzheimer's disease (Non-PatentDocuments 1 and 2).

In general, fatty acids are classified into long-chain fatty acids,medium-chain fatty acids, and short-chain fatty acids depending on thelength of the carbon chain. Medium-chain fatty acids are considered toefficiently produce ketone bodies as compared with long-chain fattyacids due to the difference in pathway of absorption/metabolism, and, inhumans and animals, medium-chain fatty acid oil and fat is known toincrease the concentration of ketone bodies in the blood as comparedwith long-chain fatty acid oil and fat (Non-Patent Document 3). However,side effects such as diarrhea and vomiting are sometimes caused byintake of medium-chain fatty acid oil and fat, which makes practicalintake of the medium-chain fatty acid oil and fat difficult (Non-PatentDocument 4).

REFERENCE LIST Non-Patent Documents

Non-Patent Document 1: Maciej Gasior, et al., Behav Pharmacol., 2006;17(5-6): p.431-439

Non-Patent Document 2: Kristin W. et al., Curr Treat Options Neurol.2008; 10(6): p.410-416

Non-Patent Document 3: Pi-Sunyer FX, et al., Diabetes., 1969; 18(2):p.96-100

Non-Patent Document 4: Henderson et al., Nutr Metalab (Lond). 2009; 6:31 doi: 10.1186/1743-7075-6-31

SUMMARY OF THE INVENTION

The present inventors have found that the ketone body production from amedium-chain fatty acid and a medium-chain fatty acid ester in a livingbody can be further promoted upon intake of a specific amino acid. Thepresent invention is based on this finding.

Namely, an object of the present invention is to provide a novelcomposition for promoting ketone body production and a novel agent forpromoting ketone body production.

The present invention provides the following inventions.

-   [1] A composition for use in promoting ketone body production and an    agent for promoting ketone body production (hereinafter referred to    as the “composition and agent according to the present invention” in    some cases), comprising, as an active ingredient, one or more amino    acid(s) selected from the group consisting of citrulline, leucine,    cysteine, taurine, glutamine and aspartic acid.-   [2] The composition and agent according to [1], which further    comprise either or both of a medium-chain fatty acid and a    medium-chain fatty acid ester.-   [3] The composition and agent according to [1] or [2], wherein the    medium-chain fatty acid ester is a medium-chain triglyceride.-   [4] The composition and agent according to [2] or [3], wherein the    molar ratio [(A)/(B)] of the amino acid(s) (A) to the medium-chain    fatty acid and medium-chain fatty acid ester (B) ranges from 0.1 to    0.5.-   [5] The composition and agent according to any one of [1] to [4],    which are each a food composition.-   [6] The composition and agent according to any one of [1] to [5],    which are for use in treating, preventing or improving a disease or    symptom that is to be effectively treated, prevented or improved by    promoting ketone body production.-   [7] The composition and agent according to [6], wherein the disease    and symptom are one or more selected from the group consisting of    childhood epilepsy, intractable epilepsy, glucose transporter 1    (GLUT1) deficiency, pyruvate dehydrogenase complex disorder,    Alzheimer's disease, neurodegenerative disease, mild cognitive    impairment, Parkinson's disease, traumatic brain injury, cancer,    depression, autism, migraine, amyotrophic lateral sclerosis,    narcolepsy, diabetes, heart failure, myocardial infarction, angina    pectoris, and obesity.-   [8] A method for promoting ketone body production and a method for    treating, preventing or improving a disease or symptom that is to be    effectively treated, prevented or improved by promoting ketone body    production, the methods comprising feeding or administering one or    more amino acid(s) selected from the group consisting of citrulline,    leucine, cysteine, taurine, glutamine and aspartic acid to a mammal.-   [9] The method for promoting ketone body production and the    treating, preventing or improving method according to [8], which    comprise feeding or administering either or both of a medium-chain    fatty acid and a medium-chain fatty acid ester in addition to the    amino acid(s).-   [10] Use of one or more amino acid(s) selected from the group    consisting of citrulline, leucine, cysteine, taurine, glutamine and    aspartic acid, for the manufacture of an agent for promoting ketone    body production or an agent for treating, preventing or improving a    disease or symptom that is to be effectively treated, prevented or    improved by promoting ketone body production.-   [11] Use of a combination of one or more amino acid(s) selected from    the group consisting of citrulline, leucine, cysteine, taurine,    glutamine and aspartic acid and either or both of a medium-chain    fatty acid and a medium-chain fatty acid ester, for the manufacture    of an agent for promoting ketone body production or an agent for    treating, preventing or improving a disease or symptom that is to be    effectively treated, prevented or improved by promoting ketone body    production.-   [12] One or more amino acid(s) selected from the group consisting of    citrulline, leucine, cysteine, taurine, glutamine and aspartic acid,    for use in the promotion of ketone body production or for use in the    treatment, prevention or improvement of a disease or symptom that is    to be effectively treated, prevented or improved by promoting ketone    body production.-   [13] A combination of one or more amino acid(s) selected from the    group consisting of citrulline, leucine, cysteine, taurine,    glutamine and aspartic acid and either or both of a medium-chain    fatty acid and a medium-chain fatty acid ester, for use in the    promotion of ketone body production or for use in the treatment,    prevention or improvement of a disease or symptom that is to be    effectively treated, prevented or improved by promoting ketone body    production.-   [14] Use of one or more amino acid(s) selected from the group    consisting of citrulline, leucine, cysteine, taurine, glutamine and    aspartic acid, as an agent for promoting ketone body production or    as an agent for treating, preventing or improving a disease or    symptom that is to be effectively treated, prevented or improved by    promoting ketone body production.-   [15] Use of a combination of one or more amino acid(s) selected from    the group consisting of citrulline, leucine, cysteine, taurine,    glutamine and aspartic acid and either or both of a medium-chain    fatty acid and a medium-chain fatty acid ester, as an agent for    promoting ketone body production or as an agent for treating,    preventing or improving a disease or symptom that is to be    effectively treated, prevented or improved by promoting ketone body    production.

The composition and agent according to the present invention can be usedto produce a sufficient amount of ketone bodies in a living body whilereducing the intake amount of a medium-chain fatty acid and amedium-chain fatty acid ester for the purpose of promoting ketone bodyproduction from the medium-chain fatty acid and medium-chain fatty acidester in a living body. Therefore, the composition and agent accordingto the present invention are advantageous in that they can promote theketone body production in a living body while reducing side effects.Further, the composition and agent according to the present inventionare advantageous in that they comprise, as an active ingredient, anamino acid which has been established to have safety, and thus can befed safely over a long term and also can realize the continuation oftreatment applied to persons who are addressing the prevention andtreatment of various diseases and the improvement in their QOL.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a reference view showing the maximum blood concentration(Cmax) and area under the blood concentration-curve (AUC) of theβ-hydroxybutyric acid concentration (bHB (mmol/L)) until the elapse of apredetermined time from an initial value (0 hours). The bloodβ-hydroxybutyric acid concentration at the initial value (0 hours)represents the concentration of β-hydroxybutyric acid originally presentin the blood before feeding of a test substance. FIG. 1B is a referenceview showing the maximum blood concentration (ΔCmax) and area under theblood concentration-curve (ΔAUC) of β-hydroxybutyric acid until theelapse of a predetermined time from an initial value (0 hours), when theconcentration of β-hydroxybutyric acid originally present in the bloodat the initial value (0 hours) before feeding of the test substance isassumed to be 0.

FIG. 2 is a graph showing the transition of the amount of change (ΔbHB)in concentration of β-hydroxybutyric acid (bHB) in the blood until 6hours after simultaneous feeding of citrulline (Cit) and medium-chainfatty acid oil and fat (MCT) for an experimental group and a controlgroup.

FIG. 3 shows the area under the blood concentration-curve (ΔAUC) ofβ-hydroxybutyric acid (% relative value for the experimental group whenthe value for the control group is assumed to be 100) until 6 hoursafter simultaneous feeding of citrulline (Cit) and medium-chain fattyacid oil and fat (MCT); t-test, *: p<0.05 vs control group.

FIG. 4 shows the maximum blood concentration (ΔCmax) of β-hydroxybutyricacid (% relative value for the experimental group when the value for thecontrol group is assumed to be 100) until 6 hours after simultaneousfeeding of citrulline (Cit) and medium-chain fatty acid oil and fat(MCT).; t-test, *: p<0.05 vs control group.

FIG. 5 is a graph showing the transition of the amount of change (ΔbHB)in concentration of β-hydroxybutyric acid (bHB) in the blood until 6hours after simultaneous feeding of leucine (Leu) and medium-chain fattyacid oil and fat (MCT) for the experimental group and the control group.

FIG. 6 shows the area under the blood concentration-curve (ΔAUC) ofβ-hydroxybutyric acid (% relative value for the experimental group whenthe value for the control group is assumed to be 100) until 6 hoursafter simultaneous feeding of leucine (Leu) and medium-chain fatty acidoil and fat (MCT).

FIG. 7 shows the maximum blood concentration (ΔCmax) of β-hydroxybutyricacid (% relative value for the experimental group when the value for thecontrol group is assumed to be 100) until 6 hours after simultaneousfeeding of leucine (Leu) and medium-chain fatty acid oil and fat (MCT);t-test, *: p<0.05 vs control group.

FIG. 8 shows the area under the blood concentration-curve (ΔAUC) ofβ-hydroxybutyric acid (% relative value for the experimental group whenthe value for the control group is assumed to be 100) until 6 hoursafter simultaneous feeding of citrulline (Cit) with varying intakeamount and medium-chain fatty acid oil and fat (MCT) in a constantamount.

FIG. 9 shows the maximum blood concentration (ΔCmax) of β-hydroxybutyricacid (% relative value for the experimental group when the value for thecontrol group is assumed to be 100) until 6 hours after simultaneousfeeding of citrulline (Cit) with varying intake amount and medium-chainfatty acid oil and fat (MCT) in a constant amount.

DETAILED DESCRIPTION OF THE INVENTION

The composition for use in promoting ketone body production according tothe present invention is characterized in that it comprises, as anactive ingredient, one or more amino acid(s) (at least one amino acid)(hereinafter referred to as the “amino acid of the present invention” insome cases) selected from the group consisting of citrulline (Cit),leucine (Leu), cysteine (Cys), taurine (Tau), glutamine (Gln) andaspartic acid (Asp).

The amino acid of the present invention may be a free body or a hydrate,and may also be a salt. Examples of the salt of the amino acid includeacid addition salts (for example, organic acid salts such as acetic acidsalts, tartaric acid salts and fatty acid salts; and inorganic acidsalts such as hydrochloric acid salts, hydrobromic acid salts, nitricacid salts, sulfuric acid salts and perchloric acid salts), salts withorganic bases (for example, organic amine addition salts such asmorpholine), and salts with inorganic bases (for example, metal saltssuch as potassium salt, sodium salt and zinc salt, and ammonium salts).The amino acid of the present invention may be any of L-bodies, D-bodiesand DL-bodies thereof. The use of L-bodies is preferred.

The amino acid of the present invention can be obtained, for example,from materials and foods containing a large amount of the amino acid, bysqueezing, concentration, purification, crystallization, extraction withvarious solvents, or the like. As such various solvents, water andcommonly-used solvents such as alcohols, hydrocarbons, organic acids,organic bases, inorganic acids, inorganic bases and supercritical fluidscan be used alone or in combination. Microbially-produced solvents,chemically-synthesized solvents or commercially-available solvents mayalso be used.

Among the amino acids of the present invention, citrulline was foundfrom watermelons. Due to its presence in a variety of living organisms,citrulline extracted (including rough extraction) or purified or roughlypurified from these living organisms or processed products thereof canbe typically used as the active ingredient of the present invention.Also, citrulline prepared by a chemical synthesis method may be used asa part or the whole of citrulline. Since citrulline is commerciallyavailable, commercially-available products may be used.

The amino acid of the present invention has the action of promotingketone body production in a living body, as will be illustrated in theExamples below. Thus, the amino acid of the present invention can beused as the active ingredient of the composition for use in promotingketone body production and the agent for promoting ketone bodyproduction, and can also be used as the active ingredient in the methodfor promoting ketone body production.

Here, the phrases “promoting ketone body production” and “promotion ofketone body production” mean that the ketone body production in a livingbody is promoted, and the degree of promotion of ketone body productioncan be evaluated, for example, by using the blood β-hydroxybutyric acidconcentration (mmol/L) as an index of the ketone body in the blood (seethe section “Evaluation of ketone body production” in the Examples). Inparticular, the degree of promotion of ketone body production can beevaluated by measuring the blood β-hydroxybutyric acid concentrationsboth when either or both of a medium-chain fatty acid and a medium-chainfatty acid ester is/are fed alone (single feeding) and when either orboth of a medium-chain fatty acid and a medium-chain fatty acid esteris/are fed in combination with the amino acid (combined feeding), andcomparing the single feeding and the combined feeding in terms of themaximum value (ΔCmax) of the amount of change in blood β-hydroxybutyricacid concentration and the area under the curve (ΔAUC). When both ΔCmaxand ΔAUC in the combined feeding take higher values than those in thesingle feeding, it can be determined that ketone body production hasbeen promoted.

Here, the term “medium-chain fatty acid” refers to saturated fatty acidshaving 6 to 12 carbon atoms. Examples of the medium-chain fatty acidinclude lauric acid, caprylic acid, capric acid, caproic acid, heptylicacid, and pelargonic acid. The term “medium-chain fatty acid ester”means esters of medium-chain fatty acids, and examples thereof includemedium-chain triglycerides (MCTs) having a structure with an ester bondbetween a medium-chain fatty acid and glycerin (for example, caprylictriglyceride and capric triglyceride). The “medium-chain triglyceride”used herein is sometimes referred to as “medium-chain fatty acid oil andfat.”

Since medium-chain fatty acids and medium-chain fatty acid esters arepresent in vegetables such as coconut and palm fruit and dairy productssuch as cow milk, medium-chain fatty acids and medium-chain fatty acidesters extracted (including rough extraction) or purified or roughlypurified therefrom can be used in the present invention. Or, productsobtained by a chemical synthesis method and commercially-availableproducts may be used as a part or the whole of the medium-chain fattyacid and medium-chain fatty acid ester.

The content of the amino acid of the present invention in thecomposition and agent according to the present invention is notparticularly limited as long as the ketone body production promotingeffect due to the amino acid of the present invention can be obtained.However, from the viewpoint of efficient feeding and administration ofthe amino acid of the present invention, the content of the amino acidin the composition and agent can be set, for example, to 1 to 20% bymass per solid content of the composition and agent, and is preferably 2to 15% by mass, more preferably 3 to 12% by mass. When two or more aminoacids of the present invention are blended in the composition and agentaccording to the present invention, the content (%) of the amino aciddefined above is the total content of the two or more amino acids.

The amino acid of the present invention promotes the ketone bodyproduction from a medium-chain fatty acid or an ester thereof in aliving body through oral feeding, as will be illustrated in the Examplesbelow. Therefore, the composition and agent according to the presentinvention are preferably fed or administered together with either orboth of the medium-chain fatty acid and the medium-chain fatty acidester.

The composition and agent according to the present invention can be fedsimultaneously with a normal food or before or after feeding thereof, orbetween meals. Examples of the food to be fed together with thecomposition and agent according to the present invention include foodscontaining an ingredient which serves as a source of ketone bodyproduction in a living body (for example, a fatty acid and a fatty acidester). From the viewpoint of better exertion of the ketone bodyproduction promoting effect due to the amino acid of the presentinvention, a food containing either or both of a medium-chain fatty acidand a medium-chain fatty acid ester is preferred.

Examples of the food containing either or both of a medium-chain fattyacid and a medium-chain fatty acid ester include, but are not limitedto, Ketonformula (manufactured by Meiji Co. Ltd.), Macton oil(manufactured by Kissei Pharmaceutical Co., Ltd.), MCT powder(manufactured by The Nissin Oillio Group, Ltd.), coconut oil and foodsadded with these foods.

When the composition and agent according to the present invention arefed or administered together with either or both of a medium-chain fattyacid and a medium-chain fatty acid ester, the composition and agent arefed so that the molar ratio [(A)/(B)] of the amino acid (A) of thepresent invention to the medium-chain fatty acid and medium-chain fattyacid ester (B) falls within the range of preferably 0.1 to 0.5, morepreferably 0.15 to 0.3, from the viewpoint of promoting the ketone bodyproduction in a living body. Also when the composition and agentaccording to the present invention are fed together with a foodcontaining either or both of a medium-chain fatty acid and amedium-chain fatty acid ester, the amount of the medium-chain fatty acidand medium-chain fatty acid ester contained in the food can bedetermined according to the above ratio. Additionally, when two or moreamino acids of the present invention are blended in the composition andagent according to the present invention, the content (mole) of theamino acid based on which the molar ratio is calculated is the totalcontent of the two or more amino acids. When either of a medium-chainfatty acid and a medium-chain fatty acid ester is fed or administered,the above molar ratio is the molar ratio of the amino acid (A) to eitherof a medium-chain fatty acid and a medium-chain fatty acid ester, ofcourse.

According to a preferred embodiment of the present invention, there isprovided a composition for use in promoting ketone body production andan agent for promoting ketone body production, further comprising eitheror both of a medium-chain fatty acid and a medium-chain fatty acid esterin addition to the amino acid of the present invention. The compositionand agent make it possible to provide the amino acid of the presentinvention having the action of promoting ketone body production in aliving body and either or both of a medium-chain fatty acid and amedium-chain fatty acid ester which serve(s) as a source of ketone bodyproduction together. Therefore, the composition and agent areadvantageous in that ketone bodies can be efficiently produced in aliving body.

The molar ratio [(A)/(B)] of the amino acid (A) of the present inventionto the medium-chain fatty acid and medium-chain fatty acid ester (B) inthe composition and agent according to the present invention rangespreferably from 0.1 to 0.5, more preferably from 0.15 to 0.3.Additionally, when two or more amino acids of the present invention areblended in the composition and agent according to the present invention,the content (mole) of the amino acid based on which the molar ratio iscalculated is the total content of the two or more amino acids. Wheneither of a medium-chain fatty acid and a medium-chain fatty acid esteris contained in the composition and agent according to the presentinvention, the above molar ratio is the molar ratio of the amino acid(A) to either of a medium-chain fatty acid and a medium-chain fatty acidester, of course.

The method for promoting ketone body production according to the presentinvention can be carried out by feeding or administering an effectiveamount of the amino acid of the present invention to a human ornon-human animal. In the method for promoting ketone body productionaccording to the present invention, as with the composition and agentaccording to the present invention, it is preferable to feed oradminister the amino acid of the present invention together with eitheror both of a medium-chain fatty acid and a medium-chain fatty acidester. From the viewpoint of efficient ketone body production in aliving body, it is more preferable to feed or administer a compositioncomprising the amino acid of the present invention and either or both ofa medium-chain fatty acid and a medium-chain fatty acid ester.

The use of the amino acid of the present invention is intended toinclude both of therapeutic use and non-therapeutic use among usesthereof in a human and a non-human animal. Here, the term“non-therapeutic” means exclusion of activities of performing anoperation, treatment or diagnosis on a human (i.e., medical activitiesto a human), and specifically means exclusion of a method of performingan operation, treatment or diagnosis on a human by a doctor or a personwho receives doctor's instructions.

The amino acid of the present invention can promote the ketone bodyproduction from the medium-chain fatty acid and medium-chain fatty acidester in a living body, as will be illustrated in the Examples below. Ithas been reported so far that childhood epilepsy and intractableepilepsy can be prevented or treated by promoting ketone body productionin a living body (Non-Patent Documents 1 and 2). It has also beenreported that the use of glucose is reduced in the brains of patientssuffering from Alzheimer's disease and mild cognitive impairment,thereby reducing the brain function and the cognitive function, and thatAlzheimer's disease and mild cognitive impairment can be prevented ortreated by ketone body production (Non-Patent Documents 1 and 2). Thus,the composition and agent according to the present invention can be usedin the treatment, prevention or improvement of a disease and a symptomthat are to be effectively treated, prevented or improved by promotingketone body production. In other words, the amino acid of the presentinvention can be used as the active ingredient of the composition foruse in the treatment, prevention and improvement of the above diseasesand symptoms and can also be used as the active ingredient of the agentsfor treating, preventing and improving the above diseases and symptoms.The amino acid of the present invention can also be used in the methodsfor treating, preventing and improving the above diseases and symptoms,and, specifically, an effective amount thereof can be administered to ahuman or non-human animal.

Examples of the diseases and symptoms that are to be effectivelytreated, prevented or improved by promoting ketone body productioninclude childhood epilepsy, intractable epilepsy, glucose transporter 1(GLUT1) deficiency, pyruvate dehydrogenase complex disorder, Alzheimer'sdisease, neurodegenerative disease such as muscular dystrophy, mildcognitive impairment, Parkinson's disease, traumatic brain injury,cancer, depression, autism, migraine, amyotrophic lateral sclerosis,narcolepsy, diabetes, heart failure, myocardial infarction, anginapectoris, and obesity (Non-Patent Documents 1 and 2 and Paoli A. et al.,Eur J Clin Nutr. 2013; 67: p.789-796).

The composition and agent for use in promoting ketone body productionaccording to the present invention can be provided in the form ofpharmaceutical products, quasi-drugs, foods, feeds, and the like, andcan be carried out according to the following description. Also, themethod for promoting ketone body production according to the presentinvention and the treating, preventing and improving methods accordingto the present invention can be carried out according to the followingdescription.

The composition and agent according to the present invention can beorally fed or orally administered, as pharmaceutical products orsupplements, to a human and a non-human animal. Examples of oral agentsinclude tablets (including sugar-coated tablets), pills, capsules,granules, powdered drugs and syrups. These formulations can beformulated by using a pharmaceutically acceptable carrier by a techniquecommonly used in the art. Examples of the pharmaceutically acceptablecarrier include excipients, binders, disintegrants, lubricants, odorimproving agents, solubilizers, suspensions, coating agents, perfumes,buffers, thickeners, colorants, stabilizers and emulsifiers. Further, anappropriate amount of a vitamin, a mineral, an organic acid, asaccharide, a peptide or the like may be added to the composition andagent according to the present invention.

In the present invention, administration to a human and a non-humananimal other than oral administration, including tube administration,nasal tube administration, drip infusion and suppositories, can also beadopted according to the form of the composition and agent according tothe present invention. For example, the composition and agent accordingto the present invention are each prepared in the form of a viscousliquid composition or a semi-solid composition, and thus can beadministered also to humans and non-human animals having deterioratedchewing and swallowing functions so that oral feeding or oraladministration cannot be adopted. By feeding or administering thecomposition and agent according to the present invention in a way otherthan oral feeding, ketone body production can be promoted in humans andnon-human animals even though their chewing and swallowing functions aredeteriorated, for example, with aging. In addition, the treatment,prevention and improvement of diseases that are to be effectivelytreated, prevented or improved by promoting ketone body production andsymptoms related to the diseases can be expected.

The composition and agent according to the present invention areprepared by using, as raw materials, ingredients contained in daily foodmaterials, such as an amino acid and a medium-chain fatty acid and amedium-chain fatty acid ester, while having the action of promotingketone body production in a living body, and thus can be provided asfoods fed daily, foods fed as supplements, and, further, functionalnutritional foods. Also, the composition and agent according to thepresent invention can be blended in various foods to be provided.

When the composition and agent according to the present invention areprovided as foods, the foods may be attached with an indication that thefoods have the action of promoting ketone body production. In this case,in order that consumers can easily understand the indication, the foodsof the present invention may be attached with an indication whichsuggests the alleviation, maintenance and improvement of diseases thatare to be effectively treated, prevented or improved by promoting ketonebody production and symptoms related to the diseases, for example,“maintaining/supporting the cognitive function.”

When the composition and agent according to the present invention areprovided as foods, the foods contain an effective amount of the aminoacid of the present invention (or the amino acid of the presentinvention and either or both of a medium-chain fatty acid and amedium-chain fatty acid ester). Here, the phrase “containing aneffective amount” of the amino acid of the present invention (or theamino acid of the present invention and either or both of a medium-chainfatty acid and a medium-chain fatty acid ester) refers to a contentwhich allows feeding of the amino acid of the present invention (or theamino acid of the present invention and either or both of a medium-chainfatty acid and a medium-chain fatty acid ester) in an amount within arange as will be described later when individual foods are fed in anormally eaten amount. The term “food” used herein includes healthfoods, functional foods, functional health foods (such as designatedhealth foods, functional nutritional foods, nutritional supplements andfoods with function claims), foods for special dietary uses (such asfoods for infants, foods for expectant and nursing mothers, foods forpatients), and medical foods (foods formulated under the control of adoctor, defined by the U.S. Food and Drug Administration (FDA) and theOrphan Drug Act). The composition and agent according to the presentinvention comprise an ingredient which can be utilized as a food, andthus can be prepared as liquid foods, oral/tube feeding nutrients,beverages, gel foods (especially, so-called functional foods) or thelike in expectation of promoting ketone body production and effectsrelated thereto, and used in the nutritional management of patients whoget nutrition orally/enterally, aged persons, babies and infants, etc.

The form of the “food” is not particularly limited, and may be a solidform such as a bar, a liquid form such as a beverage or a liquid food, apaste form, a semi-liquid form, a gel form, a powder form, or the like.Regardless of the form such as a solid form, a liquid form or a powderform, the composition and agent according to the present invention maybe added to various foods (such as confectioneries such as chocolate andice cream, cow milk, refreshing beverages, fermented milk, yogurt,cheese, bread, biscuit, cracker, pizza crust, modified milk powder,liquid foods, foods for special dietary uses, foods for patients,nutritional foods, frozen foods, processed foods, and other commercialfoods), and the foods added therewith may be fed. When the nutritionalcomposition is used in the form of a powder, the composition can bemanufactured, for example, by spray-drying, freeze-drying or any othermeans.

When the composition and agent according to the present invention areprovided as foods, the foods preferably comprise an ingredient whichserves as a source of ketone body production in a living body (forexample, either or both of a fatty acid and a fatty acid ester,preferably either or both of a medium-chain fatty acid and amedium-chain fatty acid ester) from the viewpoint of better exertion ofthe ketone body production promoting effect due to the amino acid of thepresent invention.

Examples of the food containing either or both of a medium-chain fattyacid and a medium-chain fatty acid ester include, but are not limitedto, Ketonformula (manufactured by Meiji Co. Ltd.), Macton oil(manufactured by Kissei Pharmaceutical Co., Ltd.), MCT powder(manufactured by The Nissin Oillio Group, Ltd.), coconut oil and foodsadded with these foods.

The daily intake amount or dose of the composition and agent accordingto the present invention as foods or pharmaceutical products is notparticularly limited as varying depending on the subject's pathologicalcondition, age, symptom, body weight and use and whether they are onlyfoods to be fed or pharmaceutical products to be administered fornutrition. In the case of feeding and administration for the purpose ofpromoting ketone body production in a living body and the action andeffect related thereto, it is possible to feed or administer thecomposition and agent according to the present invention, for example,so that the amino acid of the present invention can be fed in an amountof 0.2 to 10 g, preferably 0.5 to 5 g, more preferably 1 to 3 g per dayfor an adult. The intake amount and dose can also be determined by adoctor in charge of the subject. When two or more amino acids of thepresent invention are blended in the composition and agent according tothe present invention, the above effective intake amount and dose arethe total amount of the two or more amino acids.

The composition and agent according to the present invention can be fedor administered together with a food, a food additive and apharmaceutical product which effectively promote ketone body production.Examples of substances which can be fed or administered together includeMCT oil and ketone esters such as 1,3-butandiol.

The composition and agent according to the present invention can beprovided as a composition in a daily intake amount or dose which iseffective for promoting ketone body production in a living body and theaction and effect related thereto. In this case, the composition andagent according to the present invention may be packaged so as to allowfeeding of an effective daily intake amount, and the package form may becomposed of one package or a plurality of packages as long as theeffective daily intake amount can be fed. When the composition and agentaccording to the present invention are provided in a package form, it isdesirable that a description concerning the intake amount or dose begiven on the package or that a document with the description be providedtogether with the package form, in order that the subject can easilyrecognize the effective daily intake amount or dose. When the effectivedaily intake amount or dose is provided in the form of a plurality ofpackages, it is also possible to provide, as a set, a plurality ofpackages containing the effective daily intake amount or dose forconvenience of feeding or administration.

The package form for providing the composition and agent according tothe present invention is not particularly limited as long as it is aform which defines a constant amount, including package papers, bags,soft bags, paper containers, cans, bottles, capsules, and othercontainers in which the composition and agent can be contained.

The composition and agent according to the present invention arepreferably administered or fed continuously for one week or more toexert the effect better, and the period for administration and feedingis more preferably four weeks or more, particularly preferably abouttwelve weeks or more. Here, the term “continuously” means that feedingor administration is continued every day. When the composition and agentaccording to the present invention are provided in a package form,effective intake amounts or doses for a constant period (for example,four weeks) may be provided as a set for continuous feeding oradministration.

EXAMPLES

Hereinafter, the present invention will be described in more detail byway of the following examples, but is not limited thereto.

Measurement of β-hydroxybutyric Acid Concentration

In the following Examples, the blood β-hydroxybutyric acid concentrationwas measured with a β-hydroxybutyric acid measuring instrument forself-inspection (Precision Xceed manufactured by Abbott).

Evaluation of Promotion of Ketone Body Production

In the following Examples, the promotion of ketone body production wasevaluated as follows. Specifically, blood was collected from the caudalvein of rats under fasting to measure the blood concentration (mmol/L)of β-hydroxybutyric acid (bHB) as an index of the ketone body in theblood. The measured value was defined as an initial value (0 hours). Therats were grouped into a control group and an experimental group, andfed with test samples. Thereafter, the transition of the blood bHBconcentration until after 6 hours from the initial value was graphed. Areference view for determining the maximum blood concentration (Cmax)and area under the blood concentration-curve (AUC) of bHB is shown inFIG. 1A. A value obtained by subtracting the measured value at theinitial value (0 hours) from the measured value at the time of eachmeasurement was calculated, and defined as an amount of change in bloodβ-hydroxybutyric acid concentration (ΔbHB concentration) (FIG. 1B).Then, for the amount of change in blood β-hydroxybutyric acidconcentration (ΔbHB concentration), the maximum blood concentration(ΔCmax) and area under the blood concentration-curve (ΔAUC) until after6 hours from the initial value (0 hours) were calculated (FIG. 1B).

A % relative value (% ΔCmax (%)) of the maximum blood concentrationΔCmax of β-hydroxybutyric acid for the experimental group when themaximum blood concentration ΔCmax of β-hydroxybutyric acid for thecontrol group was assumed to be 100 was determined according to thefollowing calculation formula.

$\begin{matrix}{{\% \mspace{14mu} \Delta \; C\; \max \mspace{11mu} (\%)} = {\frac{\Delta \; C\; \max \mspace{14mu} {for}\mspace{14mu} {test}\mspace{14mu} {group}}{\Delta \; C\; \max \mspace{14mu} {for}\mspace{14mu} {control}\mspace{14mu} {group}} \times 100}} & \left\lbrack {{Mathematical}\mspace{14mu} {Formula}\mspace{14mu} 1} \right\rbrack\end{matrix}$

A % relative value (% ΔAUC (%)) of the area under the bloodconcentration-curve ΔAUC of blood β-hydroxybutyric acid for theexperimental group when the area under the blood concentration-curveΔAUC of blood β-hydroxybutyric acid for the control group was assumed tobe 100 was determined according to the following calculation formula.

$\begin{matrix}{{\% \mspace{14mu} \Delta \; {AUC}\mspace{11mu} (\%)} = {\frac{\Delta \; {AUC}\mspace{14mu} {for}\mspace{14mu} {test}\mspace{14mu} {group}}{\Delta \; {AUC}\mspace{14mu} {for}\mspace{14mu} {control}\mspace{14mu} {group}} \times 100}} & \left\lbrack {{Mathematical}\mspace{14mu} {Formula}\mspace{14mu} 2} \right\rbrack\end{matrix}$

Example 1 Selection of Amino Acid Promoting Ketone Body Production

Male Wistar rats (obtained from Japan SLC, Inc.) were acclimated for oneweek and then used for testing. Blood was collected from the caudal veinof the rats under overnight fasting to measure the blood concentration(mmol/L) of β-hydroxybutyric acid (bHB) as an index of the ketone bodyin the blood. The rats were grouped (n=8 for each group) so that theaverages of the body weights and the β-hydroxybutyric acidconcentrations for the respective groups were as equal as possible.Then, water was orally fed in an amount of 10 mL/kg of the body weightto the control group, and aqueous solutions of various amino acids(manufactured by Wako Pure Chemical Industries, Ltd.) were orally fed inan amount of 1.0 mmol/kg of the body weight to the experimental group.Immediately after feeding of water or each of the aqueous amino acidsolutions, caprylic triglyceride (manufactured by Tokyo ChemicalIndustry Co., Ltd.), which is medium-chain fatty acid oil and fat (MCT),was orally fed in an amount of 3.0 g/kg of the body weight to thegroups. Blood was collected from the caudal vein 1, 2, 3, 4, 5, and 6hours after feeding of MCT to measure the blood β-hydroxybutyric acidconcentration.

As a result of reviews on the various amino acids, six amino acids,i.e., citrulline (Cit), leucine (Leu), cysteine (Cys), taurine (Tau),glutamine (Gln) and aspartic acid (Asp), were selected as the aminoacids by which both the % relative value (% ΔCmax (%)) of the maximumblood concentration ΔCmax of β-hydroxybutyric acid and the relativevalue (% ΔAUC (%)) of the area under the blood concentration-curve ΔAUCof β-hydroxybutyric acid for the experimental group were higher thanthose for the control group fed with water.

The measurement results concerning the six amino acids were as follows.

TABLE 1 Measurement results concerning six amino acids % ΔCmax (%) %ΔAUC (%) Citrulline (Cit) 112.8 ± 25.5 105.4 ± 37.2 Leucine (Leu) 130.1± 31.8 110.5 ± 21.1 Cysteine (Cys) 151.9 ± 23.8 128.3 ± 22.2 Taurine(Tau) 137.8 ± 21.8 103.6 ± 23.0 Glutamine (Gln) 121.2 ± 44.6 103.6 ±30.7 Aspartic acids (Asp) 108.8 ± 23.6 107.5 ± 30.7

Example 2 Effect of Citrulline or Leucine on Ketone Body Production inFeeding of Medium-Chain Fatty Acid Oil and Fat

Male Wistar rats (obtained from Japan SLC, Inc.) were acclimated for oneweek and then used for testing. Blood was collected (0 hours) from thecaudal vein of the rats under four-hour fasting to measure theβ-hydroxybutyric acid (bHB) (mmol/L) in the blood as an index of theketone body in the blood. The rats were grouped (three groups, i.e., thecontrol group and the experimental groups; n=8 for each) so that theaverages of the body weights and the β-hydroxybutyric acidconcentrations for the respective groups were as equal as possible.Then, water was orally fed in an amount of 10 mL/kg of the body weightto the control group, and citrulline (Cit) or leucine (Leu) was orallyfed in an amount of 1.5 mmol/kg of the body weight to the experimentalgroups. Immediately after feeding of water or the aqueous citrulline orleucine solution, caprylic triglyceride (manufactured by Tokyo ChemicalIndustry Co., Ltd.) as the medium-chain fatty acid oil and fat (MCT) wasorally fed in an amount of 4.5 g/kg of the body weight to the groups.Blood was collected from the caudal vein 1, 2, 3, 4, 5, and 6 hoursafter feeding of MCT to measure the blood β-hydroxybutyric acidconcentration.

The results were as shown in FIGS. 2 to 7. From the results shown inFIGS. 2 to 4, it was confirmed that, when citrulline (Cit) was fedtogether with the medium-chain fatty acid oil and fat (MCT), the ketonebody production was promoted as compared with the case where themedium-chain fatty acid oil and fat (MCT) was fed alone. Also, from theresults shown in FIGS. 5 to 7, it was confirmed that, when leucine (Leu)was fed together with the medium-chain fatty acid oil and fat, theketone body production was promoted as compared with the case where themedium-chain fatty acid oil and fat (MCT) was fed alone.

Example 3 Effect of Citrulline Amount on Ketone Body Production inFeeding of Medium-Chain Fatty Acid Oil and Fat

Male Wistar rats (obtained from Japan SLC, Inc.) were acclimated for oneweek and then used for testing. Blood was collected (0 hours) from thecaudal vein of the rats under four-hour fasting to measure the bloodβ-hydroxybutyric acid concentration (mmol/L) as an index of the ketonebody in the blood. The rats were grouped (two groups, i.e., the controlgroup and the experimental group; n=10 for each) so that the averages ofthe body weights and the β-hydroxybutyric acid concentrations for therespective groups were as equal as possible. Then, water was orally fedin an amount of 10 ml/kg of the body weight to the control group, and anaqueous solution of citrulline was fed to the experimental group in anamount of 1.5 mmol/kg of the body weight, 2.25 mmol/kg of the bodyweight or 3.0 mmol/kg of the body weight, respectively. Immediatelyafter feeding of water or the aqueous citrulline solution, caprylictriglyceride (manufactured by Riken Vitamin Co., Ltd.) as themedium-chain fatty acid oil and fat (MCT) was orally fed in an amount of4.5 g/kg of the body weight to the groups. Blood was collected from thecaudal vein 1, 2, 3, 4, 5, and 6 hours after feeding of MCT to measurethe blood β-hydroxybutyric acid concentration. Similarly to Example 1,the maximum blood concentration (ΔCmax) and area under the bloodconcentration-curve (ΔAUC) of β-hydroxybutyric acid (bHB) until after 6hours from the initial value (0 hours) were calculated, and the %relative value (% ΔCmax (%)) of the maximum blood concentration ΔCmax ofβ-hydroxybutyric acid and the relative value (% ΔAUC (%)) of the areaunder the blood concentration-curve ΔAUC of β-hydroxybutyric acid, forthe experimental group, to those for the control group were obtained.

The results are shown in FIGS. 8 and 9. From the results shown in FIGS.8 and 9, it was confirmed that, when MCT was fed in a constant amount,the amount of the ketone bodies produced varied according to the intakeamount of citrulline (Cit), and that an optimum citrulline (Cit) dosefor promoting ketone body production was present.

1. A composition for use in promoting ketone body production,comprising, as an active ingredient, one or more amino acid(s) selectedfrom the group consisting of citrulline, leucine, cysteine, taurine,glutamine and aspartic acid and either or both of a medium-chain fattyacid and a medium-chain fatty acid ester.
 2. (canceled)
 3. Thecomposition of claim 1, wherein the medium-chain fatty acid ester is amedium-chain triglyceride.
 4. The composition of claim 1, wherein themolar ratio [(A)/(B)] of the amino acid(s) (A) to the medium-chain fattyacid and medium-chain fatty acid ester (B) ranges from 0.1 to 0.5. 5.The composition of claim 1, which is a food composition.
 6. (canceled)7. (canceled)
 8. A method for promoting ketone body production to treat,prevent, or improve a disease or symptom that can be treated, preventedor improved by promoting ketone body production in a subject in needthereof, comprising feeding or administering the the subject one or moreamino acid(s) selected from the group consisting of citrulline, leucine,cysteine, taurine, glutamine and aspartic acid in an amount effective totreat, prevent, or improve the disease or symptom.
 9. The method ofclaim 8, further comprising feeding or administering to the subjecteither or both of a medium-chain fatty acid and a medium-chain fattyacid ester in an amount effective to treat, prevent, or improve thedisease or symptom. 10.-15. (canceled)
 16. The method of claim 8,wherein the amino acid is provided in a food product.
 17. The method ofclaim 9, wherein the amino acid and either or both of the medium-chainfatty acid and medium-chain fatty acid ester are provided in a foodproduct.
 18. The method of claim 9, wherein the amino acid and the amedium-chain fatty acid and/or a medium-chain fatty acid ester areadministered in a molar ratio [(A)/(B)] of the amino acid(s) (A) to themedium-chain fatty acid and/or medium-chain fatty acid ester (B) rangesfrom 0.1 to 0.5.
 19. The method of claim 18, wherein the amino acid andeither or both of the a medium-chain fatty acid and a medium-chain fattyacid ester are provided in a food product.
 20. The method of claim 8,wherein the disease and symptom are one or more selected from the groupconsisting of childhood epilepsy, intractable epilepsy, glucosetransporter 1 (GLUT1) deficiency, pyruvate dehydrogenase complexdisorder, Alzheimer's disease, neurodegenerative disease, mild cognitiveimpairment, Parkinson's disease, traumatic brain injury, cancer,depression, autism, migraine, amyotrophic lateral sclerosis, narcolepsy,diabetes, heart failure, myocardial infarction, angina pectoris, andobesity.